The first goal of these proposed studies is to identify the fundamental cellular mechanisms by which vasoconstrictor stimuli increase pulmonary vasomotor tone. Those studies are a prerequisite to achieve our second goal, which is to elucidate the extent and the cellular mechanisms of action by which general anesthetics alter the pulmonary vascular responses to vasoconstrictor stimuli. An increase in vascular smooth muscle tone is achieved by increasing the intracellular calcium concentration ([Ca2+]i) and/or increasing myofilament Ca2+ sensitivity. We present compelling preliminary evidence that receptor-mediated, G-protein-coupled agonists that share a common signal transduction pathway (phospholipase C) have unique distal signaling mechanisms to regulate [Ca2+]i and tension in pulmonary artery smooth muscle (PASM). Our preliminary results also support the concept that anesthetic agents can alter multiple cellular mechanisms of pulmonary vasoconstriction. Aims 1 and 2 involve in vitro studies that will investigate the effects of general anesthetics, alone and in combination with agonist-induced vasoconstrictor stimuli, on cellular mechanisms that regulate PASM [Ca2+]i and myofilament Ca2+ sensitivity. Aim 3 will utilize a new methodology that we have developed to investigate the effects of anesthetics and vasoconstrictor stimuli on the intact pulmonary microcirculation. Aim 4 involves in vivo studies in chronically-instrumented dogs, and will investigate the effects of general anesthetics on the pulmonary vasoconstrictor responses to alveolar hypoxia, circulatory hypotension, and normovolemic hemodilution. The proposed studies will utilize a variety of experimental preparations, including: 1) isolated sarcoplasmic reticulum (SR) vesicles to directly measure PASM SR Ca2+ uptake, release, and content; 2) purified myofibrils to directly measure PASM actomyosin ATPase activity; 3) PASM cells to measure [Ca 2+]i, membrane potential, ion currents, inositol phosphate production and phosphorylation of contractile proteins; 4) Western blot analysis to measure protein tyrosine phosphorylation and immunofluorescent techniques to measure translocation of protein kinase C isoforms; 5) PASM strips to simultaneously measure changes in [Ca2+]i and tension, as well as intracellular pH; 6) intravital microscopy to directly measure pulmonary microvascular diameter; and 7) continuous left pulmonary vascular pressure-flow plots in chronically-instrumented dogs in the conscious and anesthetized states. The proposed studies are unique in that we will combine in vitro, microcirculatory, and in vivo approaches. We believe that these studies will yield fundamental information about cellular mechanisms of pulmonary vascular regulation, which should provide insight about mechanisms of pulmonary vascular disease. Moreover, our results will elucidate cellular mechanisms of anesthetic action, which should provide insight concerning the optimal choice of anesthetic agent to minimize increases in right ventricular "afterload" in patients with right ventricular dysfunction or failure.